{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T02:36:41Z","timestamp":1784774201166,"version":"3.55.0"},"reference-count":79,"publisher":"Wiley","issue":"9","license":[{"start":{"date-parts":[[2024,9,4]],"date-time":"2024-09-04T00:00:00Z","timestamp":1725408000000},"content-version":"vor","delay-in-days":3,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2024,9]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The sexually transmitted pathogen, <jats:italic>Neisseria gonorrhoeae<\/jats:italic>, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and the panmictic structure of the gonococcal population. However, high\u2010frequency transformation also makes <jats:italic>N. gonorrhoeae<\/jats:italic> one of the easiest bacterial species to manipulate genetically in the laboratory. Techniques have been developed that result in transformation frequencies &gt;50%, allowing the identification of mutants by screening and without selection. Constructs have been created to take advantage of this high\u2010frequency transformation, facilitating genetic mutation, complementation, and heterologous gene expression. Similar methods have been developed for <jats:italic>N. meningitidis<\/jats:italic> and nonpathogenic <jats:italic>Neisseria<\/jats:italic> including <jats:italic>N. mucosa<\/jats:italic> and <jats:italic>N. musculi<\/jats:italic>. Techniques are described for genetic manipulation of <jats:italic>N. gonorrhoeae<\/jats:italic> and commensal <jats:italic>Neisseria<\/jats:italic> species, as well as for growth of these fastidious organisms. \u00a9 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Spot transformation of <jats:italic>Neisseria gonorrhoeae<\/jats:italic> on agar plates<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Spot transformation of commensal <jats:italic>Neisseria<\/jats:italic> on agar plates<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Transformation of <jats:italic>Neisseria gonorrhoeae<\/jats:italic> in liquid culture<\/jats:p><jats:p><jats:bold>Basic Protocol 4<\/jats:bold>: Electroporation of <jats:italic>Neisseria gonorrhoeae<\/jats:italic><\/jats:p><jats:p><jats:bold>Basic Protocol 5<\/jats:bold>: Creation of unmarked mutations using a positive and negative selection cassette<\/jats:p><jats:p><jats:bold>Basic Protocol 6<\/jats:bold>: In vitro mutagenesis of <jats:italic>Neisseria gonorrhoeae<\/jats:italic> chromosomal DNA using EZ\u2010Tn5<\/jats:p><jats:p><jats:bold>Basic Protocol 7<\/jats:bold>: Chemical mutagenesis<\/jats:p><jats:p><jats:bold>Basic Protocol 8<\/jats:bold>: Complementation on the <jats:italic>Neisseria gonorrhoeae<\/jats:italic> chromosome<\/jats:p><jats:p><jats:bold>Alternate Protocol 1<\/jats:bold>: Complementation with replicating plasmids<\/jats:p><jats:p><jats:bold>Alternate Protocol 2<\/jats:bold>: Complementation on the <jats:italic>Neisseria musculi<\/jats:italic> or <jats:italic>Neisseria mucosa<\/jats:italic> chromosome<\/jats:p><jats:p><jats:bold>Basic Protocol 9<\/jats:bold>: Preparation of chromosomal DNA from <jats:italic>Neisseria gonorrhoeae<\/jats:italic> grown on solid medium<\/jats:p><jats:p><jats:bold>Alternate Protocol 3<\/jats:bold>: Preparation of chromosomal DNA from <jats:italic>Neisseria gonorrhoeae<\/jats:italic> grown in broth<\/jats:p><jats:p><jats:bold>Support Protocol<\/jats:bold>: Preparing PCR templates from <jats:italic>Neisseria gonorrhoeae<\/jats:italic> colonies<\/jats:p>","DOI":"10.1002\/cpz1.70000","type":"journal-article","created":{"date-parts":[[2024,9,4]],"date-time":"2024-09-04T07:31:37Z","timestamp":1725435097000},"source":"Crossref","is-referenced-by-count":12,"title":["Genetic Manipulation of <i>Neisseria gonorrhoeae<\/i> and Commensal <i>Neisseria<\/i> Species"],"prefix":"10.1002","volume":"4","author":[{"given":"Joseph P.","family":"Dillard","sequence":"first","affiliation":[{"name":"Medical Microbiology and Immunology University of Wisconsin\u2010Madison  Madison Wisconsin"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jia Mun","family":"Chan","sequence":"additional","affiliation":[{"name":"Division of Infection and Immunity University College London  London"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2024,9,4]]},"reference":[{"key":"e_1_2_19_2_1_1","doi-asserted-by":"publisher","DOI":"10.1128\/JB.01408\u201006"},{"key":"e_1_2_19_2_2_1","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00171\u201013"},{"key":"e_1_2_19_2_3_1","doi-asserted-by":"publisher","DOI":"10.1128\/jb.129.2.983\u2010992.1977"},{"key":"e_1_2_19_2_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0378\u20101119(97)00653\u20107"},{"key":"e_1_2_19_2_5_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142727.mb0302s13"},{"key":"e_1_2_19_2_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/0378\u20101119(93)90695\u2010Y"},{"key":"e_1_2_19_2_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/0378\u20101119(94)00890\u20105"},{"key":"e_1_2_19_2_8_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1175653"},{"key":"e_1_2_19_2_9_1","doi-asserted-by":"publisher","DOI":"10.1128\/jb.160.1.288\u2010293.1984"},{"key":"e_1_2_19_2_10_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00792\u2010014\u20100663\u20108"},{"key":"e_1_2_19_2_11_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1218832110"},{"key":"e_1_2_19_2_12_1","volume-title":"Generation and release of inflammatory peptidoglycan fragments by pathogenic and nonpathogenic Neisseria","author":"Chan J. 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